Brucellosis is a worldwide zoonosis of considerable social and economic importance. It is considered by the Food and Agriculture Organisation (FAO) and World Health Organisation (WHO) to be the most widespread zoonosis in the world, and is on the Office International des Epizooties (OIE) List B, denoting diseases of significant socio-economic, public health and international trade importance. The causative agents of the disease are facultative intracellular bacteria of the monospecific genus Brucella melitensis. Despite extensive genetic homogeneity amongst strains, historical classification defines six separate species, B. melitensis, B. abortus, B. suis, B. ovis, B. canis and B. neotomae, and a multitude of phenotypically defined sub-species or biovars (reviewed in Verger et al., 1987, Ann. Inst. Pasteur Microbiol. 138: 235-238). This nomenclature remains common practice amongst researchers in the field, and has epidemiological and clinical relevance. Brucella infections can be found in a wide range of animal hosts. The six species display distinct animal host preferences and have variable virulence in man. B. abortus infections are mostly associated with the disease in cattle, B. melitensis with small ruminants and camelids, B. suis with pigs, hares, rodents and rangifers (depending on biovar), B. ovis with sheep and B. canis with dogs. B. neotomae has only ever been reported in desert wood rats.
Diagnosis of Brucella infection can be achieved via Complement Fixation Test, Serum Agglutination Test, Rose Bengal antigen Test, and ELISA (Enzyme-Linked Immunosorbent Assay) methods. Routine surveillance for brucellosis is carried out in several countries using a simple indirect ELISA method. The antigen used in the ELISA is a crude preparation of Brucella LPS. The LPS of Brucella is the serodominant antigen associated with smooth strain infections of Brucella. 
A number of vaccines are currently licensed for use in livestock against the various important pathogenic species of Brucella, all of which are live attenuated strains of the organism. B. abortus S19 and B. abortus RB51 vaccines have been used for the protection of cattle against brucellosis (for review see Cheville, 2000, Ann. NY Acad. Sci. 916: 147-153). S19 was developed through laboratory culture and selection of altered phenotypic characteristics when compared to the parent strain B. abortus 544. RB51 was similarly selected as a stable ‘rough’ mutant of B. abortus 2308. B. suis S2, a naturally occurring avirulent isolate of B. suis biovar 1, has been used extensively for the vaccination of pigs in China, where success rates are reportedly high. However, evaluation of this vaccine in other locations has resulted in extremely variable efficacy and its use has not been widely adopted.
The live attenuated vaccine B. melitensis Rev.1 has been established as recommended for the protection of sheep and goats against B. melitensis (Alton, 1987, Trop. Anim. Health Prod. 19: 65-74; Garin-Bastuji et al., 1998, Vet. Res. 29: 255-274) and is intermittently used as part of the control strategy in affected Mediterranean countries. Rev.1 was derived through laboratory culture and selection of altered phenotypic characteristics compared to the parent strain B. melitensis. There are currently no Brucella vaccines available for the prophylaxis of human infection.
The use of a live attenuated vaccine strain essentially involves the deliberate infection of the naïve animal with attenuated Brucella such that protective immune responses are elicited without concurrent pathology. The genetic mechanism underlying the attenuation of each of the vaccine strains is not fully understood.
Although there have been attempts to produce non-living vaccines, these have not been as widely accepted or successful as the live strains. One of the most widely studied non-living vaccines was the B. abortus 45/20 vaccine. An adjuvanted bacterin vaccine was reported to be protective in cattle and did not directly cause abortion. However, yearly boosters were required to maintain immunity, and local reactogenicity at the inoculation site was found to be a considerable problem. Furthermore, insufficient standardization in the production and evaluation methods for the killed preparations resulted in unacceptable levels of variability in efficacy between batches. Ultimately the use of the 45/20-adjuvant vaccine was abandoned in favor of the more consistent and longer-lived immunizing properties of the live vaccines such as S19. Numerous other attempts to generate non-living Brucella vaccines, either using simple bacterins or fractionated antigen preparations, have concluded that these preparations are poorly immunogenic.
Similarly, a number of recombinant protein based vaccines have been assessed but in each case these have been unable to promote comparable protective immunity to the live vaccines. A variety of delivery strategies have been employed for these recombinant antigens ranging from simple direct inoculation of the protein to formulation with adjuvants such as CpG ODN or delivery via vaccinia or Escherichia coli. 
Recently a novel non-living vaccine based upon antigenic extracts of rough strain B. ovis has been described (Murillo et al., 2001, Vaccine 19: 4099-5106). The vaccine consists of hot saline extracted membrane antigens microencapsulated within poly-ε-caprolactone particles, and is able to protect rams against infection with B. ovis. However, at present there are not any non-living vaccines that are licensed for use against brucellosis, and none with reported efficacy against smooth strain Brucella. 
We have previously reported on the development and in vitro assessment of five DNA vaccines encoding the genes omp25, FliC, FrpB, AcvB and invasion protein B (“iaIB”, also known as “invB” and “iaiB”) (Commander et al., 2003, Poster presentation at “Brucellosis 2003” Conference, Pamplona, Spain, September 15-17). It was demonstrated that mice receiving a naked DNA vaccine based upon either iaIB or omp25 were able to control infection by B. melitensis 16M to a similar level to those receiving the live attenuated vaccine Rev.1. The protective effect of the vaccines was seen using four intramuscular inoculations of 100 μg plasmid, given at three week intervals.
Numerous mechanical and formulation strategies have been investigated for ability to increase in vivo transfection efficiencies. Among the widely reported mechanical strategies for improving the delivery of DNA vaccine plasmids to host cells are ballistic devises referred to “Gene guns”, and in vivo electroporation strategies. In addition to the mechanical strategies, the physical formulation of a vaccine can impact upon immunogenicity and efficacy. The formulation of vaccines with classical adjuvants such as alum, and oil and water emulsions are the most notable examples of this.
Vaccines such as B. abortus S19 and B. melitensis Rev.1 are live attenuated smooth strain infections and therefore induce LPS specific immune responses that cannot readily be distinguished from the immune response associated with a virulent infection. Differentiation of animals producing antibodies in response to LPS from vaccine strains or virulent strains is not possible using LPS-detection assays such as ELISA assays, resulting in vaccinated animals presenting as ‘Brucella positive’ in serodiagnostic tests. The practical and political issues of differential diagnosis are an impediment to an effective vaccination-based control strategy. Since Brucella reactors have to be eliminated in order to maintain or obtain Officially Brucellosis Free (“OBF”) status, prophylactic vaccination using for example the approved Rev.1 vaccine is incompatible with test and slaughter campaigns.